We investigated gut microbial diversity profiles and their intra-species variations within several populations of Australian native, eastern grey Kangaroo. Three hypervariable regions of the 16S rRNA gene (V1-V3, V3-V4, and V5-V6) were analysed from faecal samples collected from 30 Eastern Grey Kangaroos (Macropus giganteus) across six locations of South-East Queensland. Two methods were used for obtaining the faecal microbial genomic DNA. In the first method, DNA extraction was carried out on composite samples of equal amounts of faecal material of five kangaroos, whereas in the second method the microbial genomic DNA was extracted from each individual kangaroo first and equal amounts of DNA from each kangaroo were pooled into 6 composite samples (five kangaroos each). Both methods used the QiaAmp Powerfaecal DNA extraction kit (Qiagen) for faecal microbial DNA extraction. We obtained 150bp paired-end reads using the Illumina Miseq platform and constructed microbial taxonomic profiles for each variable region. Composite samples containing genomic DNA extracted from individual faecal samples yielded greater intraspecies variability than combining faeces material prior to DNA extraction. Diversity profiling of gut bacteria using V1-V3 hypervariable region yielded a broader range of taxa due to its longer target region. Higher levels of unassigned taxa were obtained with the V1-V3 regions, these unassigned taxa decreased ≥ 5-fold when analysed using the V3-V4 and V5-V6 regions, which enabled classification of the unassigned into identifiable bacterial taxa. There was insufficient evidence to suggest which hypervariable regions contain the greatest intra-species diversity as each target hypervariable region demonstrated sequence diversity among different bacteria.